Oncogenes—genes that contribute to the production of cancers—are generally mutated forms of certain normal cellular genes (“proto-oncogenes”). Oncogenes often encode abnormal versions of signal pathway components, such as receptor tyrosine kinases, serine-threonine kinases, or downstream signaling molecules. The central downstream signaling molecules are the Ras proteins, which are anchored on the inner surfaces of cytoplasmic membranes, and which hydrolyze bound guanosine triphosphate (GTP) to guanosine diphosphate (GDP). When activated by a growth factor, growth factor receptors initiate a chain of reactions that leads to the activation of guanine nucleotide exchange activity on Ras. Ras alternates between an active “on” state with a bound GTP (hereafter “Ras.GTP”) and an inactive “off” state with a bound GDP. The active “on” state, Ras.GTP, binds to and activates proteins that control the growth and differentiation of cells.
For example, in the “mitogen-activated protein kinase (MAP kinase) cascade,” Ras.GTP leads to the activation of a cascade of serine/threonine kinases. One of several groups of kinases known to require a Ras.GTP for their own activation is the Raf family. The Raf proteins activate “MEK1” and “MEK2,” abbreviations for mitogen-activated ERK-activating kinases (where ERK is extracellular signal-regulated protein kinase, another designation for MAPK). MEK1 and MEK2 are dual-function serine/threonine and tyrosine protein kinases and are also known as MAP kinase kinases. Thus, Ras.GTP activates Raf, which activates MEK1 and MEK2, which activate MAP kinase (MAPK). Activation of MAP kinase by mitogens appears to be essential for proliferation, and constitutive activation of this kinase is sufficient to induce cellular transformation. Blockade of downstream Ras signaling, as by use of a dominant negative Raf-1 protein, can completely inhibit mitogenesis, whether induced from cell surface receptors or from oncogenic Ras mutants.
The interaction of Raf and Ras is a key regulatory step in the control of cell proliferation. To date, no substrates of MEK other than MAPK have been identified; however, recent reports indicate that MEK may also be activated by other upstream signal proteins such as MEK kinase or MEKK1 and PKC. Activated MAPK translocates and accumulates in the nucleus, where it can phosphorylate and activate transcription factors such as Elk-1 and Sap1a, leading to the enhanced expression of genes such as that for c-fos.
Once activated, Raf and other kinases phosphorylate MEK on two neighboring serine residues, S218 and S222 in the case of MEK-1. These phosphorylations are required for activation of MEK as a kinase. In turn, MEK phosphorylates MAP kinase on two residues separated by a single amino acid: a tyrosine, Y185, and a threonine, T183. MEK appears to associate strongly with MAP kinase prior to phosphorylating it, suggesting that phosphorylation of MAP kinase by MEK may require a prior strong interaction between the two proteins. Two factors—MEK's unusual specificity and its requirement for a strong interaction with MAP kinase prior to phosphorylation—suggest that MEK's mechanism of action may differ sufficiently from the mechanisms of other protein kinases as to allow for selective inhibitors of MEK. Possibly, such inhibitors would operate through allosteric mechanisms rather than through the more usual mechanism involving blockage of an ATP binding site.
MEK1 and MEK2 are, therefore, validated and accepted targets for anti-proliferative therapies, even when the oncogenic mutation does not affect MEK structure or expression. See, e.g., U.S. Patent Publications 2003/0149015 by Barrett et al. and 2004/0029898 by Boyle et al.
An N-unsubstituted, 3-hydroxy-5-phenyoxyphenyl-isothiazole-4-carboxyamidine having anti MEK activity was described in a patent publication commonly assigned with the present application. US 2004/0039037 (Feb. 26, 2004). No analogs or derivatives of the compound were described. A group of 3-phenyloxy-5-alkylureido-isothiazole-4-carboxamide kinase inhibitors having anti-proliferative activity was reported in U.S. Pat. No. 6,548,526 and No. 6,235,764. These compounds were not noted to be MEK inhibitors; they inhibited a tyrosine kinase activity associated with the vascular endothelial growth factor receptor (VEGF).
A second group of isothiazole-4-carboxamides with anti-proliferative activity via blockage of Trk tyrosine kinase—not a VEGF-associated kinase—was described in U.S. Patent Publication 2004/0152691. The compounds are 3-arylsulfanyl-5-heteroarylamino-isothiazoles.
Isoxazole inhibitors of MEK-activated kinase (ERK or MAPK) were described in U.S. Pat. No. 6,649,640 and No. 6,495,582. Rather than 3-hydroxy-4-carboxamides or 3-hydroxy-4-carboxamidines, the compounds disclosed in these patents are 3-unsubstituted or 3-amino substituted-4-aryl isoxazoles.